Role of C-reactive Protein and Tumor Necrosis Factor-Alpha in Differentiating between Ventilator-Associated Pneumonia and Systemic Inflammatory Response Syndrome without Infectious Etiology.

BACKGROUND
Differential diagnosis of systemic inflammatory response syndrome (SIRS) with or without infectious cause is critically important in terms of initiating antimicrobial agents in case of infectious etiology such as ventilator-associated pneumonia (VAP). The aim of this study was to determine the diagnostic and prognostic roles of C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α) in differentiating between ventilator-associated pneumonia and SIRS without infectious etiology.


MATERIALS AND METHODS
In this prospective observational study, 91 adult intensive care unit (ICU) patients were enrolled. According to established diagnostic criteria, they were classified into three groups of "non-SIRS non-VAP", "SIRS non-VAP" and "SIRS-VAP". Serum CRP and TNF-α were measured on days 1, 3 and 7 of the study and compared using repeated measures ANOVA.


RESULTS
With respect to diagnosis, there was no significant difference in the values of these biomarkers between groups (P>0.05). There was no statistically significant "time trend" for C-reactive protein and TNF-α (P>0.05). Considering both group effect and Time effect, the changes were not significantly different for CRP (P= 0.86) and TNF-α (P=0.69). In contrast, the clinical score and the clinical pulmonary infection score (CPIS) ≥ 6, had 100% specificity for diagnosing VAP. With respect to prognosis, only an unchanged or decreasing TNF-α from day 1 to day 3 was marginally associated with 28-day survival. However, day 1 and day 3 acute physiology and chronic health evaluation II (APACHE II) scores were highly associated with 28-day survival.


CONCLUSION
Unlike clinical scoring system including CPIS and APACHE II, TNF-α and CRP levels were not useful as diagnostic or prognostic biomarkers for differentiating between SIRS with VAP etiology and SIRS without infectious etiology.


INTRODUCTION
Biomarker measurement in critically ill patients has received increasing attention (1,2). Biomarkers can aid in diagnosis or prognosis (1). For example, in a clinical ICU setting the measurement of CRP is now included in the surviving sepsis campaign international guidelines as a reported to be prognostic of outcome (1). For example, elevated CRP levels have been reported to be prognostic of increased mortality in critically ill septic patients (4,5). Too frequently, these studies omit the comparison of the measured value of biomarker to the value of existing clinical scoring systems. Thus, it is often unclear whether biomarker measurement adds to clinical management and, in particular, if biomarker measurement is most helpful in establishing a diagnosis or by improving prognostic estimates.
To address these issues, we chose to study a common yet challenging problem in the ICU -the diagnosis of VAP.
VAP is a very common cause of morbidity and mortality in ICU patients (2,6,7). The clinical diagnosis of VAP is usually based on systemic signs of infection, new or expanding pulmonary infiltrates seen on chest roentgenogram and bacteriologic evidence of pulmonary parenchymal infection (6). Although, microbiologic diagnosis of VAP is crucial for specific diagnosis and management, it takes a substantial period of time to obtain culture results.
Thus, VAP is an important ICU problem that would greatly benefit from enhanced diagnostic capacity provided by rapid biomarker measurements (8)(9)(10).
CRP measurement helps the diagnosis of infection (11).
However, specificity for infection has been raised as a limitation (12). CRP is an acute-phase protein produced by the liver, the levels of which rise in response to  and APACHE II scores were determined (23).

CRP and TNF-α measurements
Blood samples were drawn on the first, third and seventh study days for CRP and TNF-α measurements.
Blood samples were collected in glass tubes and placed in ice containers. Samples were processed within two hours by centrifuging at 1,600 g for 15 minutes. Plasma supernatant was rapidly frozen and preserved at -70 o C until final analysis. CRP concentration was measured using a particle enhanced turbidimetris assay (Roche, Germany) and TNF-α concentration was measured using an enzymelinked immune sorbent assay (ELISA. eBioscience, Austria).

Statistical analysis
We  Table 1.

Biomarkers as diagnostic factors
There were no significant differences in the values of CRP and TNF-α between groups (between-subject differences or group effect) (Figure 1). In addition, there was no statistically significant time trend (within-subject differences or time effect) for CRP and TNF-α ( Figure 1).

Biomarkers as prognostic factors
Two, eight and 17 patients died by days three, seven and 28 after ICU admission, respectively. We tested early biomarker levels (day 1 and day 3) for prognostic value of mortality, reasoning that many deaths had occurred by day 7 and day 7 was late in the typical course of VAP.
Biomarker levels (median and range) in survivors and nonsurvivors (28 days after study inclusion) on day one and day three are shown in Table 3. While CRP and TNF-α were not predictive of mortality, APACHE II scores were significantly different between survivors and nonsurvivors on day one (P=0.008) and day three (P=0.005). With regard to prognosis we found that clinical scoring using the APACHE II score was a highly significant predictor of 28-day mortality. In contrast, neither CRP nor TNF-α were predictive of 28-day mortality. In further analysis, we found that no change or reduction in concentration of TNF-α from day 1 to day 3 was more common in survivors than non-survivors. Hillas et al, also demonstrated that higher level of CRP at day 7 in VAP patients was associated with development of septic shock, although it could not predict VAP survival (24).
The sensitivity of CRP on day 1 to discriminate "SIRSnon-VAP" from "SIRS-VAP" was very low (e.g., 33.3%). In most previous studies, CRP levels have been referred to be an indicator of morbidity and mortality rather than a diagnostic test (10,11,17,25,26). Póvoa et al. found that in community acquired sepsis patients admitted to the ICU, the survivors had a lower CRP level on days three to five of stay in the ICU compared to non-survivors (11). The pattern of CRP changes could predict the postoperative complications and higher one-year mortality in patients undergoing esophagectomy (10). CRP levels more than 10 mg/dL were associated with 6.6 times higher mortality in respiratory ICU patients (25). Considering the prognostic utility of CRP, it was used successfully for assessing response to antibiotics (8), risk stratification of cardiovascular disease (17) and for predicting acute brain dysfunction in critically-ill patients (26).
In cardiac surgery patients, serum CRP was not a diagnostic marker for VAP although procalcitonin was (27). Similar results of preference of procalcitonin over CRP was reported in discriminating SIRS and sepsis (18) and also as a marker for detection of early VAP (9) or VAP in patients with a successful cardiopulmonary resuscitation (28). The assay of CRP in bronchoalveolar lavage fluid was also not helpful in the diagnosis of VAP (29). Decrease in CRP, as decrease in PCT, Sequential Organ Failure Assessment and APACHE II was associated with the prediction of survival of VAP patients (30). APACHE II is an established and feasible outcome predictor tool in critically-ill patients including septic patients who are at high risk of death and who are more likely to benefit from intervention (31,32). We found that APACHE II is a better predictor of survival compared to CRP and TNF-α.
In our study, TNF-α, similar to CRP, could not differentiate between "SIRS-VAP" and "SIRS-non-VAP", although higher TNF-α on day three was associated with a higher mortality. The sensitivity of TNF-α at all three time points was more than 70%, but the area under the curves of sensitivity and specificity was not significantly different from 0.5. In most studies, the trend of inflammatory mediators was compared between SIRS and sepsis/septic shock (19,33). A higher level of TNF-α was observed in sepsis patients compared to SIRS or control groups. Also, higher TNF-α level was associated with higher occurrence of disseminated intravascular coagulation and mortality. In contrast, it was reported that TNF-α dynamics were not associated with risk estimation of mortality in SIRS of infectious origin (19). In our study, we found a prognostic role for TNF-α at D3, as higher level was inversely associated with survival, irrespective of the primary cause of inflammation.
Recently, simultaneous use of several inflammatory markers including cell-surface (e.g., triggering receptor expressed by myeloid cells-1, CD11b and CD62L) and soluble markers (IL-1beta, IL-6, IL-8, sTREM-1, Procalcitonin) has been used successfully to discriminate between VAP and non-VAP cases (34). One limitation of our study was that we did not measure some other biomarkers such as IL-6 and IL-8 in our study.
The overall conclusion of the study is that TNF-α and CRP levels were not capable of differentiating "SIRS-non-VAP" from "SIRS-VAP" patients. Instead, a readily available diagnostic scoring system, CPIS  6, had 100% specificity for diagnosing VAP. Similarly, TNF-α and CRP levels had little prognostic ability while a standard clinical severity of illness scoring system, APACHE II, was significantly prognostic. These prospective and carefully timed measurements raise doubt as to whether current biomarkers add substantially to current clinical practice in VAP diagnosis and prognosis.